There have been considerable efforts to identify substances that potentiate an immune response to a vaccine. For example, the cytokine interleukin-12 (IL-12) was reported to be useful as an adjuvant in U.S. Pat. No. 5,976,539, where the IL-12 was provided as a protein or nucleotide coding sequence. Interferon alpha is another cytokine reported to be useful as an oral vaccine adjuvant in, e.g., U.S. Pat. No. 4,820,514. In this patent only the protein form of the cytokine was used. Furthermore, oral administration of the protein resulted in a systemic effect, and too low or too high a dose of the protein was undesirable. It would be advantageous to generate a local adjuvant effect that does not require oral route administration of the protein and is not so dependent upon dose level.
An important aspect of the development of an adjuvant is the two-fold breadth potential for its application. Such breadth concerns both the underlying immune system and the target of the immune system's defense. An adjuvant can be useful in many mammalian species, if the adjuvant has the capacity to influence common attributes of the immune systems of those species. Further, if the targets of the immune system analogously have common attributes in how they are susceptible to immune responses, then an adjuvant stimulating an immune response should have broad applicability in defending against a variety of targets.
A need is recognized in the art for enhanced veterinary vaccines. An area of significant need for enhanced vaccines is manifested by outbreaks of porcine reproductive and respiratory syndrome (PRRS). PRRS has a severe impact on the health and reproductive ability of swine. To address this problem, vaccines were developed for the etiological agent of PRRS as reported in U.S. Pat. Nos. 5,989,563 and 6,042,830. In these patents, a viral agent capable of causing PRRS disease was isolated and used to develop a modified-live virus (MLV) vaccine. Classically, modified-live virus vaccines are derived from virulent strains that are modified by techniques such as growth in vitro, with or without alteration of conventional in vitro conditions, passage in non-natural host cells, or a combination of those techniques. In the U.S. Pat. Nos. 5,989,563 and 6,042,830 patents, the reported parental virulent porcine viral isolate is ATCC-VR2332. From this isolate, a modified vaccine virus was generated by attenuation through multiple passages of growth in vitro in simian cell culture. Thus ATCC-VR2495 was reported to be a suitable MLV for use in vaccine formulations for commercial purposes.
Currently the PRRS vaccines, even those that are of the MLV type, do not confer adequate protection from disease. In a study designed to test the efficacy of PRRS vaccines, currently available products failed to completely protect pigs from clinical disease caused by certain virulent strains of PRRS virus. Osorio et al., 1998. It was speculated that deficiencies in the pig's ability to mount cellular immune responses to PRRS virus may promote the evolution of more virulent strains in addition to predisposing the animal to virus infection. Alternatively, it was suggested that the PRRS virus itself may be responsible for altering the cellular immune response. Osorio et al., 1998.
Although the mechanisms that mediate protective immunity against PRRS virus are unknown, attempts have been made to study the characteristics of immunity induced by either infection with wild-type PRRS virus or vaccination with a commonly used PRRS modified live virus (MLV) vaccine. An example of PRRS MLV is described in U.S. Pat. Nos. 5,989,563 and 6,042,830. Exposure of an animal to either the wild-type virus or the MLV form of the PRRS virus does not stimulate a strong viral purging immunity. Virus-specific T cells secreting interferon gamma and virus neutralizing antibodies, both of which have the potential to mediate viral purging, are detected only several weeks after exposure of pigs to PRRS virus. One possible explanation for the failure of PRRS virus to stimulate the development of a strong viral purging immunity is that PRRS virus, in contrast to other viruses, is a poor stimulator of interferon alpha (abbreviated IFNalpha or IFNα) production in swine. Albina et al., 1998. Buddaert et al., 1998. Van Reeth et al., 1999. Consistent with this explanation is a reference indicating that IFNα can affect the development of anti-viral T cell interferon gamma (IFNgamma or IFNγ) responses and peak anti-viral immune defenses. Cousens et al., 1999.
There is considerable evidence in the art, however, that mere augmentation of IFNα in conjunction with wild-type PRRS virus infection or PRRS MLV exposure would be expected to have no significant positive effect on an immune response. For example, while the importance of IFNα on IFNγ response was shown in the mouse, it is unknown whether a similar response would occur in the pig. If the level of IFNα in the pig is indeed unnecessary for the induction of an adequate IFNγ response, then additional IFNα present in the pig would have no effect on immune response. Furthermore, while it is believed that PRRS virus can avoid the effects of the interferon system by blocking the production of IFN or by inhibiting its effects, it is not known which method of avoidance, blocking production or inhibition of effects, is significant or whether both methods function in a given organism. Albina, 1998. The lack of understanding about how PRRS directly or indirectly affects the innate IFNα response also makes it unclear that any attempts to further induce endogenous IFNα, to add IFNα (exogenous), or to add IFNα cDNA to express IFNα would enhance the immune response to PRRS. For example, if PRRS alters an IFNα response by inhibition of IFNα expressed protein, then it would be expected that no attempt to boost the level of IFNα would succeed in enhancing the immune response to PRRS. In fact, until now it has appeared unlikely that boosting the level of IFNα in combination with PRRS MLV exposure would have any clear effect on enhanced immunity. The present invention makes the unexpected discovery that boosting the level of IFNα in an animal can indeed yield enhanced immunity.
The fact that IFNα can enhance immunity coupled with any live virus vaccine is particularly surprising in light of the normal function of IFNα. Since IFNα is known to have a negative effect on the process of viral replication, one might reasonably expect that an immune response from exposure to a live vaccine virus would be negatively affected by a boosted level of IFNα.
Even more strikingly, evidence indicates that a heightened level of IFNα could in fact contribute to a disease state in an infected or exposed animal. For example, the proinflammatory cytokines IFNα, tumor necrosis factor alpha, and interleukin-1 have been shown to play key roles in several respiratory disease conditions. Van Reeth, 2000. From studies involving various porcine virus infections, it was proposed that the relatively low IFNα response following PRRS infection is related to the lack of acute respiratory disease, severe lung necrosis, and inflammation. Van Reeth, 1999. According to Van Reeth, the absence of a particular ‘cytokine combination’ such as IFNα, TNFa, and IL-1 during PRRS infection may in part explain the mild respiratory pathology and the absence of respiratory disease. As a corollary to this proposal, the augmentation of IFNα exogenously or endogenously, is predicted to contribute to a more severe disease state rather than to enhance immunity.
Prior research also reveals that at least three other cytokines, IFNγ, IL-12, and IL-18, share the capacity of type one interferons (including IFNalpha and IFNbeta) to augment immunity by inducing strong T cell proliferation under in vivo conditions. Sprent et al., 2000. Given that “the mechanisms involved here are still unknown,” it is unexpected that a single cytokine such as IFNα serves to enhance an in vivo immune response, if there are multiple independent pathways for achieving a similar result. Currently, “the biological significance” of at least one type of immune enhancement, T-cell proliferation, “induced by type 1 interferons and other cytokines in vivo is still unclear.” Sprent et al., 2000.
Non-human animal interferons are described in both protein form, U.S. Pat. No. 5,831,023, and in cDNA form (cDNA that expresses IFNα), U.S. Pat. No. 5,827,694. While these references disclose potential use of IFNα protein, including IFNα expressed from cDNA, in pharmaceutical compositions for prophylactic or therapeutic treatment of non-human animals, the particular use of IFNα in combination with a live virus vaccine is not disclosed and would be disfavored by those skilled in the art for the reasons discussed herein.
Substances capable of inducing endogenous interferon have been identified. Levine, 1970. These substances include live viruses with either DNA or RNA genomes, double stranded RNA, DNA from protozoan parasites, bacterial endotoxin, mannan, mitogens (phytohemagglutinin, streptolysin O, and poke-week mitogen), statolon, helenine, and synthetic polyribonucleotides.
Other inducers of interferon have been identified. In U.S. Pat. No. 5,730,971, the substances flavin adenine dinucleotide, flavin adenine mononucleotide, and riboflavin (vitamin B2) are disclosed as potential contributors to the potentiation of an interferon response.
More recently, an adjuvant role for certain short bacterial immunostimulatory DNA sequences was proposed due to their ability to stimulate T helper-1 responses in animals vaccinated with genetic versions of antigens. Roman M et al., 1997, Nature Med 3:849. These DNA sequences are known to contain CpG (Cytosine-Guanine) motifs that are believed to be significant in the immunostimulatory capacity. However, such sequences are suggested for use in vaccine compositions where the vaccine component or subunit thereof is inactivated and not for use with live organisms such as viruses or modified live viruses.
Inducers of interferon were reported as having potential for resisting viral infection and for treating viral diseases as disclosed in U.S. Pat. Nos. 4,124,702 and 4,389,395. In U.S. Pat. No. 4,124,702, complexes of polymers are reported for induction of interferon production. The polymers can be synthetic homopolynucleotides such as polyriboinosinic acid and polyribocytidylic acid mixed in a 1:1 molar ratio (polyIC). For example, a modified polyIC complex was reported to induce serum interferon in primates in a fashion superior to polyIC. Levy, 1975. In U.S. Pat. No. 4,389,395, complexes comprising polyIC, poly-L-lysine and carboxymethylcellulose are reported for use in the induction of endogenous interferon; these complexes are referred to as polyICLC. Both polyIC and polyICLC were assessed for their ability to induce interferon responses in pigs. Loewen, 1986; Loewen, 1988. Jordan, 1995. It is believed that the polyIC and polyICLC provide preferential enhancement of cell-mediated versus humoral immunity. Alternatively, the benefits of polyIC and polyICLC is believed to be the result of quantitative improvement, for example in increases of the numbers of activated cells or the amount of antibody produced. These and other possible non-exclusive mechanisms are ways that interferon inducers can aid in immunity.
The present invention provides methods and compositions that enhance the efficacy of vaccines, particularly modified live viruses (MLV). In particular, the invention enhances the immunity in pigs compared with the immunity achieved by vaccination with PRRS MLV alone. The ability to enhance immunity is shown by combining a vaccine with any of three adjuvants: IFNα protein, IFNα cDNA, and inducers of endogenous IFNα production.